垂体瘤转化基因( PTTG) 的研究进展

 1997年LinPei等为了证明垂体肿瘤细胞中mRNA有差异性表达，利用差异显示PCR、North-ernblot、DNA测序等分子生物学技术发现，有一条约396bp的DNA片段与当时分离出的基因库中已知序列无同源性，用此片段从垂体瘤细胞中检测到一个长约1．3kb的mRNA呈高表达，而在正常垂体细胞中则没有。为进一步描述这个垂体肿瘤mRNA的特征，他们用从大鼠垂体瘤细胞系GH4中分离出的mRNA构建了一个cDNA文库，用396bpDNA片段作探针，从cDNA文库中分离出一条长约974bp的cDNA克隆，这个cDNA被命名为PTTG（pituitary tumor transforming gene）。随后，从人T细胞淋巴瘤瘤细胞系Jurkat细胞，人胚胎肝细胞中克隆出了大鼠PTTG 的相应物[ 3 ] ,称为人PTTG(hPTTG) 。更进一步研究证明hPTTG是一个至少含有3 个成员的基因家族[ 4 ,5 ] 。 广泛的研究显示PTTG 不仅表达在多种肿瘤中,而且在体内高增殖性的组织中(如睾丸,胸腺) 亦有高度的活性,这提示PTTG在肿瘤的发生中有十分重要的作用。     

运用原位杂交和二步法技术检测乳腺癌PTTG基因表达

目的　研究PTTG基因在乳腺癌组织中表达的临床意义。方法　应用原位杂交和二步法 ,检测 66例乳腺癌、2 7例乳腺小叶增生及 5 4例乳腺正常组织中PTTGmRNA和PTTG蛋白的表达。结果　PTTGmRNA在乳腺癌、乳腺小叶增生和乳腺正常组织中表达阳性率分别为 71.2 % (4 7/66)、5 1.9% (14 /2 7)和 11.1% (6/5 4) ,而PTTG蛋白表达阳性率分别为 63 .6% (4 2 /66)、48.1% (13 /2 7)和 7.4% (4 /5 4)。乳腺癌、乳腺小叶增生与乳腺正常组织之间的PTTGmRNA及PTTG蛋白阳性率比较均有显著性差异 (P <0 .0 5 ) ;PTTGmRNA的表达与PTTG蛋白表达呈正相关 (P <0 .0 5 ) ,而与淋巴结转移无关 (P >0 .0 5 )。结论　PTTG基因的过度表达可能参与了人乳腺癌的发生发展过程     

PTTG和p53的表达与大肠癌淋巴结转移的关系

[目的]探讨垂体瘤转化基因(PTTG)和p53蛋白在大肠癌中的表达及其淋巴结转移的关系。[方法]采用免疫组织化学法检测71例大肠癌标本和42例癌旁正常黏膜组织的PTTG和p53蛋白的表达水平,并分析其与临床病理学特征的关系。[结果]PTTG蛋白和p53在大肠癌组织中的过度表达率分别为73.2%和70.4%,均明显高于其在正常大肠黏膜组织中的表达(7.1%和0),差异具有显著性(P<0.01)。PTTG的过度表达与淋巴结转移明显相关(P<0.001),p53的过度表达则与淋巴结转移不相关(P>0.05)。[结论]PTTG在大肠癌组织中呈过度表达,并与淋巴结转移有关,可以作为判断大肠癌是否有淋巴结转移的分子指标。     

Estrogen Receptor Levels and Tumor Growth in a Series of Pituitary Clonal Cell Lines in Rats

Four kinds of in vitro clonal pituitary tumor cell lines named MtT/Se, MtT/SM, MtT/S and MtT/ E, each of which shows different sensitivity to estrogen on proliferation, were inoculated into fat pad of ovariectomized rats and estrogen-loaded ovariectomized rats at 10⁵ and 10⁶ cells/site. They formed tumor with average latency ranging from 30 to 71 in ovariectomized rats and 13 to 63 days in estrogenized rats inoculated with 106 cells. MtT/Se was highly sensitive to estrogen for growth, and MtT/SM also grew well in estrogenized rats. With MtT/S and MtT/E, there was no significant shortening of average tumor latency in estrogenized rats. In vivo , the cytosolic estrogen receptor (ER) levels of MtT/Se, SM, S and E were measured to be 452 ±66, 370 ±115, 260 ±16 and 83 ±8 fmol/ mg protein, respectively. In vitro , however, the lowest ER level was noted in MtT/Se. Histologically, all four tumors grown in rats were composed of homogeneous round cells, and MtT/Se contained particularly large nucleated cells. In MtT/E, the cells appeared to be changing into ftbromatous cells. Three cell lines except MtT/E maintained the function of hormonal secretion in vivo as well as in vitro . Serum GH level was increased in rats with MtT/Se and MtT/S. Increased levels of both prolactin and growth hormone were measured in sera of rats with MtT/SM. Increases of hormones as well as tumor sizes were promoted by the estrogen.     

Inhibitory Effect of Tamoxifen on Diethylstilbestrol-promoted Hepatic Tumorigenesis in Male Rats and Its Possible Mechanism of Action

Male Sprague-Dawley rats were given a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg body weight). Two weeks later the rats were divided into 4 groups; DEN-C group rats were given no further treatment; DEN-DES group rats were fed diethylstilbestrol (DES, 0.5 mg/day); DEN-TMX group rats were given tamoxifen (TMX, 1.0 mg/ day) orally; DEN-DES TMX group rats were fed both DES and TMX for 8 months. Rats of the DEN-DES group developed grossly visible hepatic tumors. On the other hand, tumor development was significantly inhibited in rats of the DEN-DES TMX group. Total area of γ-glutamyl transpeptidase-positive lesions and the mean area per lesion were significantly larger in rats of the DEN-DES group than those of the DEN-C, DEN-TMX or DEN-DES TMX group. Estrogen receptor (ER) content of liver cytosol assayed by enzyme immunoassay (EIA) was significantly greater in rats of the DEN-DES group than in those of the DEN-C group and smaller in rats of the DEN-TMX and DEN-DES TMX group than in the DEN-C group. On the contrary, ER content of liver nuclei was significantly greater in rats of the DEN-TMX and DEN-DES TMX group than in those of the DEN-C or DEN-DES group. These results suggest that the promotive action of DES and the inhibitory action of TMX on DES-promoted hepatic tumorigenesis are, at least in part, mediated by ER in the rat.     

Strain Difference of Susceptibility to 4-Nitroquinoline 1-Oxide-induced Tongue Carcinoma in Rats

Strain difference of susceptibility to 4-nitroquinoline 1-oxide (4NQO)-induced squamous cell carcinomas of the tongue among Dark-Agouti, Long-Evans, Sprague-Dawley, ACI/Ms, Fischer 344, Donryu and Wistar/Furth rats was surveyed by evaluating the survival times, incidences and sizes of developed tumors as markers of susceptibility. Administration of 4NQO dissolved in drinking water induced squamous cell carcinomas in various sites of the upper digestive tract mucosa of all the experimental male and female rats of the seven strains. Regarding the mean survival times, Wistar/Furth rats survived much longer than any other strain of rats, and Dark-Agouti showed the shortest survival. The incidence of large, mass-type carcinomas of the tongue of Dark-Agouti rats was higher than in any other strain of rats, while that of Wistar/Furth rats was the lowest. Subsequently the mitotic activity and bromodeoxyuridine incorporation in the tongue epithelium of Dark-Agouti and Wistar/Furth rats were estimated after a short-term administration of 4NQO. There was a pronounced difference between the two strains of rats, because the proliferative responses of the tongue epithelium of Dark-Agouti rats to the 4NQO stimulation were much higher than those of Wistar/Furth rats. These results indicated that there are marked differences in the susceptibility to 4NQO-induced tongue carcinoma among the seven strains of rats, and that Dark-Agouti and Wistar/Furth rats could be useful as models of highly and poorly susceptible strains, respectively, for further genetic analysis.     

Stimulatory Effects of Retinoic Acid on Tumor Growth and Serum Insulin-like Growth Factor-1 in Rats Bearing Estrogen-responsive Pituitary Tumor MtT/Se

MtT/Se is one of 4 cell lines derived from an estrogen-dependent pituitary tumor, MtT/F84. The main difference between these tumor types is that MtT/F84 secretes both growth hormone (GH) and prolactin (PRL) whereas MtT/Se secretes only GH. MtT/Se grew slowly in ovariectomized (ovex) rats, but tumor growth was much faster in estrogen-treated ovex rats. Effects of dietary retinoic acid (RA) on tumor growth, serum GH and insulin-like growth factor-1 (IGF-1) levels were examined in ovex rats. Latency of tumor growth was shortened, and tumor take and weight were promoted by all- trans RA both in the presence and absence of exogenous estrogen. Serum GH and IGF-1 levels became increased in tumor-hearing rats whereas PRL levels remained unchanged. Serum IGF-1 levels exhibited a good correlation with tumor weights ( r =0.84). Our results suggest a close relationship between increase of tumor weight and stimulation of serum IGF-1 level by RA in tumor-bearing rats.     

Strain Differences in N-Butyl-N-(4-hydroxybutyl)nitrosamine Bladder Carcinogenesis in Rats

Differences in susceptibility of the urinary bladder epithelium to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in various strains were examined. In experiment 1, 5 strains of male rats were given 0.025% BBN in the drinking water for 8 weeks followed by drinking water without BBN for 32 weeks. Analbuminemic rats (NAR) and ACI rats had high incidences of urinary bladder lesions (papillary or nodular hyperplasia, papilloma and carcinoma), F344 and Wistar rats had low incidences, and Sprague-Dawley (SD) rats showed an intermediate incidence. Carcinoma area was largest in NAR rats followed in decreasing order by SD, ACI and F344 rats. The extent of tumor invasion was higher in NAR and ACI rats than in SD rats. In experiment 2, the 5 strains of male rats were administered 0.025% BBN in the drinking water. Some rats from each group were killed after each of weeks 4 and 8. The urinary bladder of ACI and NAR rats given BBN had the most marked lesions observed by scanning electron microscopy, with less marked changes in SD rats. F344 and Wistar rats showed the weakest response. Cytochrome P-450 content of the liver in ACI rats treated with BBN for 4 weeks was significantly higher than those of the controls. Cytochrome P-450 and Cytochrome b ₅ contents of the control and BBN-treated rats were significantly higher in ACI and SD rats than in Wistar, F344 or NAR rats. These results indicate that there are strain differences in the urinary bladder response to BBN.     

实时荧光定量PCR检测结直肠癌hPTTG1的表达及其意义

背景与目的:虽然人垂体瘤转化基因1(human pituitary-tumortransforming gene 1,hPTTG1)在结直肠癌等恶性肿瘤中高表达,然而hPTTG1mRNA与结直肠癌临床病理参数之间的关系及其能否作为结直肠癌诊断和预后判断的分子标志尚不清楚.本研究检测hPTTG1 mRNA在结直肠癌中的表达.分析其与临床病理参数之间的关系,探讨hPTTG1 mRNA作为结直肠癌诊断和转移复发的分子标志的可能性.方法:采用实时荧光定量PCR检测结直肠癌组织与对应癌旁组织hPTTG1 mRNA,并分析其与临床病理指标的关系.结果:结直肠癌组织hPTTG1mRNA表达较止常癌旁组织显著增高(0.42±0.07 vs.0.03±0.01.P<0.001).hPTG1 mRNA与肿瘤大小、血清CEA水平有关,肿瘤直径≥3.5 cm较<3.5 cm显著增高(15.80±8.80 vs.10.91±5.22.P<0.05).血清CEA>5 ng/mL较<5ng/mL显著增高(22.79±7.42 vs.9.39±2.61,P<0.001).hPTTG1 mRNA在Dukes'C、D期显著高于Dukes'A、B期(15.88±8.09、25.69±7.67 vs, 9.03±0.35、9.58±2.93,P<0.001).伴有淋巴转移、肝转移或其他器官转移者(17.63±8.47、31.07±4.10和22.78±6.39)较无转移者(11.15±6.65)显著增高(P<0.001).hPTTG1 mRNA与患者的性别、年龄和病理分型无关(P>0.05).结论:hPTTG1 mRNA在结直肠癌中高表达.与结直肠癌的疾病进展密切相关,有助于对患者病情进展的预测.     

CD44v9基因在恶性卵巢肿瘤中的表达及其临床意义

目的：探讨恶性缲ＣＤ４４Ｖ８基因表达及其临床意义。方法：用ＲＴ－ＰＣＲ方法检测了４１例恶性卵巢肿瘤、２０例良性卵巢肿瘤和２１例正常卵巢组织中ＣＤ４４Ｖ９基因表达情况并分析相关的临床病理因素。结果：１）ＣＤ４４Ｖ９基因表达阳性纺恶性组明显高于良性组和正常组,良性组高于正常组：２）ＣＤ４４Ｖ９基因表达在有转移者阳性率显著高于无转移者,转移灶阳性率高于原发灶：３）ＣＤ４４Ｖ９阳性表达率与临床期别有一定关     

表皮生长因子对胶质瘤C6细胞垂体瘤转化基因影响的实验研究

目的探讨表皮生长因子（EGF）在体外对胶质瘤C6细胞垂体瘤转化基因（PTTG）表达的影响。方法不同浓度的EGF（10ng／mL、20ng／mL和30ng／mL）在体外作用于胶质瘤C6细胞,半定量逆转录聚合酶链式反应（RT—PCR）检测PTTG mRNA表达,Western检测其PTTG蛋白表达。结果与空白对照组比较,RT—PCR和Western检测结果显示PTTG mRNA及其蛋白的表达在各EGF作用组均显著增高,各组间比较差异有统计学意义（P〈0.01）。结论EGF可以量效的方式上调PTTG的表达。     

Inhibitory Effects of Antioxidants on N-Bis(2-hydroxypropyl)nitrosamine-induced Lung Carcinogenesis in Rats

Potential second-stage modifying effects of 8 antioxidants on lung tumorigenesis initiated by N-bis(2-hydroxypropyl)nitrosamine (DHPN) were examined in male F344 rats. After an initial 2-week treatment with DHPN (0.1% in drinking water), rats were administered one of the antioxidants supplemented in the diet for 30 weeks. Although the incidences of lung adenomas were not affected, those of carcinomas were lowered by 2% butylated hydroxyanisole (BHA, 2 rats/20 rats), 1% butylated hydroxytoluene (BHT, 1/20), 0.8% ethoxyquin (EQ, 3/20) and 1%α-tocopherol (α-TP, 2/20) treatments as compared to the control level (9/20), while 5% sodium l -ascorbate (SA), 0.8% catechol (CC), 0.8% resorcinol (RN), and 0.8% hydroquinone (HQ) did not exert any significant effect on incidence. Quantitative analysis of adenomas and carcinomas (numbers and areas of lesions per unit area of lung section) revealed obvious inhibitory effects of SA, CC, and RN as well as BHA, BHT, EQ, and α-TP. Among the antioxidants, BHT exerted the strongest inhibitory activity. In contrast, DHPN-induced thyroid tumorigenesis was significantly enhanced by BHT (14/20) and EQ (20/20) treatments (control=5/20). Thus the antioxidants showed opposite effects on lung and thyroid carcinogenesis in the rat.     

Expression of Pituitary Tumor Transforming Gene 1 is an Independent Factor of Poor Prognosis in Localized or Locally Advanced Prostate Cancer Cases Receiving Hormone Therapy

We investigated the prognostic value of pituitary tumor transforming gene 1 (PTTG1) expression according toclinicopathological features among localized or locally advanced prostate cancer cases receiving hormone therapy.A retrospective study involved 64 patients receiving combined androgen blockade treatment was performed.PTTG1 expression was determined by immunohistochemical staining using initial needle biopsy specimensfor diagnosis. Associations of PTTG1 with various clinicopathological features and disease-free survival wereexamined via uni- and multivariate analyses. No association between PTTG1 expression and clinical T stage,Gleason score, pretreatment PSA levels, risk groups was found (p =0.682, 0.184, 0.487, 0.571, respectively).Univariate analysis revealed that increased PTTG1 expression, T3 stage and high risk group were associatedwith increased risk of disease progression (p =0.000, 0.042, and 0.001), and high PSA level had a tendency topredict disease progression (p =0.056). Cox hazard ratio analysis showed that PTTG1 low expression (p =0.002),PTTG1 high expression (p =0.000) and high risk group (p =0.0147) were significantly related to decreased diseasefreesurvival. In conclusion, PTTG1 expression determined by immunohistochemical staining in needle biopsyspecimens for diagnosis is a negative prognostic factor for progression in localized or locally advanced prostatecancer receiving hormone therapy.     

Translocation t(12;16)(q13;pll) in Myxoid Liposarcoma of a Child and Implica of the Human int-1 Gene in Tumorigenesis

Cytogenetic and gene analyses were performed in a child with myxoid liposarcoma (MLS). A reciprocal chromosome translocation t(12;16)(q13:pll) was found in the tumor cells. This result, combined with the previous reports of a similar translocation in adult MLS strongly suggests that this translocation may be a characteristic cytogenetic marker in MLS. The human int -1 gene has been reported to be located close to the 12q1 breakpoint associated with MLS. Therefore, we examined the rearrangement of the human int -1 gene by Southern blotting analysis. When genomic DNAs from the tumor cells were digested with Kpn I, Eco RI and BamHI, no difference was seen compared to peripheral blood leukocytes (PBL) DNAs from a normal individual. However, with Hin dIII digestion there appeared a 3.1 kb fragment in tumor cell DNA as compared to a 2.8 kb fragment in DNAs prepared from normal PBL and the patient's PBL. These findings suggest that the int -1 gene may be implicated in tnmorigenesis of MLS with t(12;16)(q13;pll).     

β-Catenin (Ctnnb1) Gene Mutations in Diethylnitrosamine (DEN)-induced Liver Tumors in Male F344 Rats

Alterations in multiple phosphorylation sites on exon 3 of the β-catenin gene have recently been implicated in hepatocarcinogenesis in humans as well as mice. To identify genetic alterations which could be involved in the chemical-induced hepatocarcinogenesis of rats, we analyzed the status of the sites in the β-catenin gene (Ctnnb1) of liver neoplasms induced by diethylnitrosamine (DEN) in male F344 rats, using the polymerase chain reaction-single strand conformation polymorphism method. In the present investigation, we examined 35 hepatocellular neoplasms (28 adenomas and 7 carcinomas) for the expression of mutations in the region of the β-catenin gene. Point mutation at codon 32, 35, 37 or 41, which has been reported in human and mouse liver cell carcinomas and/or other cancers, was recognized in eleven (31%) out of 35 lesions (8 adenomas and 3 carcinomas). Our results indicate that Ctnnb1 mutations may contribute to hepatocarcinogenesis in rats. Our finding that Ctnnb1 mutation was present in adenomas as well as carcinomas also suggests that the mutation is a relatively early event in DEN-induced hepatocarcinogenesis in rats.     

Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is induced by genistein through the expression of p53 in colorectal cancer cells

Genistein is an isoflavenoid found in soy that has anti-tumorigenic activities. Treatment of colorectal carcinoma HCT-116 cells with 50 microM genistein results in a 50% reduction in cell proliferation and a 6-fold increase in apoptosis. Genistein induces nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1), a protein with antitumorigenic activities, in a time- and concentration-dependent manner in HCT-116 cells. In addition, p53 and p21 are induced in HCT-116 cells. The induction of p53 (3 hr) precedes the induction of NAG-1 (12 hr), suggesting that genistein-induced NAG-1 expression is mediated by p53. In contrast, NAG-1 is not induced by genistein in the p53-negative colorectal carcinoma cell line HCT-15. Luciferase reporter constructs of the NAG-1 promoter containing 2 p53 sites showed that the p53 sites within the NAG-1 promoter are critical to genistein-induced NAG-1 expression in p53-positive U2OS cells. The expression of p53 was critical for NAG-1 promoter activity since no promoter activity was observed with genistein treatment in HCT-15 cells. However, genistein-induced promoter activity was restored in HCT-15 cells by transfection with wild-type p53. Together our data suggest a relationship between genistein, p53 and NAG-1 forming a novel pathway responsible for the antitumorigenic activity of genistein.     

Lung tumorigenesis induced by human vascular endothelial growth factor (hVEGF)-A165 overexpression in transgenic mice and amelioration of tumor formation by miR-16

Many studies have shown that vascular endothelial growth factor (VEGF), especially the human VEGF-A₁₆₅ (hVEGF-A₁₆₅) isoform, is a key proangiogenic factor that is overexpressed in lung cancer. We generated transgenic mice that overexpresses hVEGF-A₁₆₅ in lung-specific Clara cells to investigate the development of pulmonary adenocarcinoma. In this study, three transgenic mouse strains were produced by pronuclear microinjection, and Southern blot analysis indicated similar patterns of the foreign gene within the genomes of the transgenic founder mice and their offspring. Accordingly, hVegf-A₁₆₅ mRNA was expressed specifically in the lung tissue of the transgenic mice. Histopathological examination of the lung tissues of the transgenic mice showed that hVEGF-A₁₆₅ overexpression induced bronchial inflammation, fibrosis, cysts, and adenoma. Pathological section and magnetic resonance imaging (MRI) analyses demonstrated a positive correlation between the development of pulmonary cancer and hVEGF expression levels, which were determined by immunohistochemistry, qRT-PCR, and western blot analyses. Gene expression profiling by cDNA microarray revealed a set of up-regulated genes (hvegf-A₁₆₅, cyclin b1, cdc2, egfr, mmp9, nrp-1, and kdr) in VEGF tumors compared with wild-type lung tissues. In addition, overexpressing hVEGF-A₁₆₅ in Clara cells increases CD105, fibrogenic genes (collagen α1, α-SMA, TGF-β1, and TIMP1), and inflammatory cytokines (IL-1, IL-6, and TNF-α) in the lungs of hVEGF-A₁₆₅-overexpressing transgenic mice as compared to wild-type mice. We further demonstrated that the intranasal administration of microRNA-16 (miR-16) inhibited lung tumor growth by suppressing VEGF expression via the intrinsic and extrinsic apoptotic pathways. In conclusion, hVEGF-A₁₆₅ transgenic mice exhibited complex alterations in gene expression and tumorigenesis and may be a relevant model for studying VEGF-targeted therapies in lung adenocarcinoma.     

肾细胞癌肿瘤抑制基因VHL的 变异多药耐药基因Mdr的表达

 目的　探讨人肾细胞癌中的VHL肿瘤抑制基因的变异和Mdr基因的表达水平，同肾癌发生发展的关系。方法　采用多聚酶链式反应(PCR)，DNA单链多态性分析和DNA测序的方法分析了40例肾细胞癌标本(其中包括30例富糖原型，6例是嗜染型、3例多形性、1例颗粒型)。用免疫组织化学法检测了正常肾组织和35例肾细胞癌组织切片。结果　19/30(63%)的富糖原型肾细胞癌的VHL肿瘤抑制基因发生了变异，其中包括13例缺失、3例插入、2例错译和1例无义。而在对照组未发现基因变异。而Mdr表达与组织学类型相关，且表达量与肿瘤分级有关。结论　富糖原型肾细胞癌可能在分子遗传学水平上与其它类型的肾细胞癌有所区别。